Bacterial lipopolysaccharide (LPS) is a potent stimulator of the cells of the immune system, including macrophages, B cells and endothelial cells (EC). Vascular EC activation or injury by LPS is considered critical in the development of endotoxic shock and blood brain barrier disruption in gram-negative meningitis. While the cellular and molecular interactions between LPS and macrophages have been studied, such interactions between LPS and EC are not well elucidated. The investigator and others have shown that LPS recognition systems between EC and macrophages are different. The intracellular signaling pathways of LPS have been largely derived from monocytes/macrophages and have only been partially characterized. LPS signaling mechanisms in EC are unknown. The proposed experiments are targeted to investigate intracellular signaling pathways induced by LPS in EC. The applicant's group has recently isolated and immortalized human brain microvessel EC (HBMEC) with SV40-Large T. Using primary and transfected HBMEC they have observed that LPS rapidly induces the tyrosine phosphorylation of several EC proteins in a sCD14-dependent manner. They identified three of the LPS-induced phosphoproteins in HBMEC as ERK1, ERK2 and p38 MAP kinase isoforms. They observed that a herbimycin A-sensitive step(s), presumably a tyrosine kinase, is required for LPS-induced IL-6 release from HBMEC. The proposed experiments will further elucidate the role of protein phosphorylation and dephosphorylation in LPS-induced IL-6 production in HBMEC. They will determine the role of MAP kinase activation in LPS-induced IL-6 release from HBMEC by determining the effects of overexpressing wild type and kinase-dead mutants of ERK1 and ERK2 (dominant-negative mutants), and the recently cloned MAP kinase phosphatases 1 and 2, on the biological and biochemical responses of HBMEC. They will next examine the signaling events upstream of MAP kinase, specifically the role of the protein kinase Raf-1, and GTP-binding protein Ras by genetic and biochemical methods. They will determine whether LPS activates c-Raf-1 and Ras in HBMEC. We will determine if Raf-1 and/or Ras activation is required for LPS-induced IL-6 release in HBMEC by determining the effect of overexpressing wild type and dominant-negative mutants on the responses to LPS. Characterization of the network of intracellular signals that result in the activation of vascular EC stimulated by LPS could provide information leading to novel approaches for the prevention and therapy of Gram negative sepsis and meningitis, where LPS-mediated EC activation has been shown to be detrimental.